Enzymatic Modification of Cell-Surface H Antigen by Glycosyltransferases

Abstract

This invention relates to cells with modified blood group antigen expression of the ABO group phenotype. It relates to the enzymatic modification of cell-surface H antigen, by the addition of one or more monosaccharide units generating cells which are serologically equivalent to A or B antigen red blood cells and uses thereof in haematology, immuno-haematology and immunology assays as serology controls.

1 . A serology control including cells comprising a derivative of an H-antigen molecule wherein the cell surface epitope of the antigen has been modified.  
   
   
       2 . A serology control according to  claim 1  where the cell surface epitope has been enzymatically modified.  
   
   
       3 . A serology control according to  claim 1  where the cell surface epitope is modified by the attachment of one or more monosaccharide units.  
   
   
       4 . A serology control according to  claim 3  where the monosaccharide units are selected from the group including galactose and N-acetylgalactosamine.  
   
   
       5 . A serology control according to  claim 4  where the monosaccharide units are alpha-linked.  
   
   
       6 . A serology control according to  claim 5  where the modified cell surface epitope is serologically equivalent to the cell surface epitope of A-antigen.  
   
   
       7 . A serology control according to  claim 5  where the derivative is serologically equivalent to A-antigen.  
   
   
       8 . A serology control according to  claim 5  where the modified cell surface epitope is serologically equivalent to the cell surface epitope of B-antigen.  
   
   
       9 . A serology control according to  claim 5  where the derivative is serologically equivalent to B-antigen.  
   
   
       10 . A serology control according to  claim 1  where the level of expression of modified cell surface epitope or derivative is serologically equivalent to the level of expression of A-/B-antigen by cells of a weak or poorly expressing ABO subgroup.  
   
   
       11 . A serology control according to  claim 1  where the level of expression of modified cell surface epitope or derivative is serologically equivalent to the clinically significant threshold for expression of A-/B-antigen by cells of an ABO blood group phenotype.  
   
   
       12 . A serology control according to  claim 1  where the cell is a red blood cell.  
   
   
       13 . A serology control according to  claim 12  where the cell is a human red blood cell.  
   
   
       14 . A serology control according to  claim 1  where the level of expression of modified cell surface epitope or derivative is less than 5×10 5  copies per cell, or the serological equivalent thereof.  
   
   
       15 . A serology control according to  claim 1  where the level of expression of modified cell surface epitope or derivative is less than 1×10 5  copies per cell, or the serological equivalent thereof.  
   
   
       16 . A serology control according to  claim 1  where the level of expression of modified cell surface epitope or derivative is less than 2×10 4  copies per cell, or the serological equivalent thereof.  
   
   
       17 . A serology control according to  claim 1  where the Level of expression of modified cell surface epitope or derivative is greater than 1×10 2  copies per cell, or the serological equivalent thereof.  
   
   
       18 . A serology control according to  claim 1  where the level of expression of modified cell surface epitope or derivative is more than 1×10 3  copies per cell, or the serological equivalent thereof.  
   
   
       19 . A serology control according to  claim 1  where the modified cell surface epitope is serologically equivalent to the antigenic determinant of an immunodominant sugar.  
   
   
       20 . A serology control according to  claim 19  where the modified cell surface epitope is serologically equivalent to the immunodominant sugar of an A-antigen or B-antigen.  
   
   
       21 . A method of preparing a serology control by modifying the cell surface epitopes of a cell comprising the steps of: 
 a. contacting a solution of immunodominant sugar modifying enzyme and a solution of activated monosaccharide units with a suspension of the cells; and    b. maintaining the suspension obtained at a temperature and for a time sufficient to allow modification of the cell surface epitope.    
   
   
       22 . A method of preparing a serology control by modifying the cell surface epitope of an H-antigen molecule according to  claim 21  comprising the steps of: 
 a. contacting a solution of immunodominant sugar modifying enzyme and a solution of activated monosaccharide units with a suspension of H-antigen expressing cells; and    b. maintaining the suspension obtained at a temperature and for a time sufficient to allow modification of the cell surface epitope of the H-antigen.    
   
   
       23 . A method according to  claim 21  where the modification of the cell surface epitope is by glycosylation.  
   
   
       24 . A method according to  claim 21  where the immunodominant sugar modifying enzyme is a glycosyltransferase.  
   
   
       25 . A method according to  claim 24  where the glycosyltransferase is an alpha-N-acetylgalactosaminyl transferase or alpha galactosyl transferase or a mixture of both.  
   
   
       26 . A method according to  claim 21  where the activated monosaccharide units are UDP-galactose, UDP-N-acetylgalactosamine, or a mixture of both.  
   
   
       27 . A method according to  claim 21  where the method provides a cell comprised of a derivative of an H-antigen wherein the cell surface epitope of the antigen has been modified and is serologically equivalent to the epitope of an A- or B-antigen.  
   
   
       28 . A method according to  claim 21  where the method provides a cell comprised of a derivative of an H-antigen molecule wherein the derivative is serologically equivalent to an A- or B-antigen.  
   
   
       29 . A method according to  claim 21  where the activity of immunodominant sugar modifying enzyme is limiting for the rate of the modification.  
   
   
       30 . A method according to  claim 21  where the concentration of activated monosaccharide units is limiting for the rate of the modification.  
   
   
       31 . A method according to  claim 21  where the method includes the step of terminating the modification reaction.  
   
   
       32 . A method according to  claim 31  where the step of terminating the modification reaction is by washing of the suspension obtained following maintaining the suspension at a temperature and for a time sufficient to allow modification.  
   
   
       33 . A serology control prepared by the method according to  claim 21 .  
   
   
       34 . A serology control according to  claim 33  where the cell surface epitope has been enzymatically modified.  
   
   
       35 . A serology control according to  claim 33  where the cell surface epitope is modified by the attachment of one or more monosaccharide units.  
   
   
       36 . A serology control according to  claim 33  where the monosaccharide units are selected from the group including galactose and N-acetylgalactosamine.  
   
   
       37 . A serology control according to  claim 33  where the monosaccharide units are alpha-linked.  
   
   
       38 . A serology control according to  claim 33  where the modified cell surface epitope is serologically equivalent to the cell surface epitope of A-antigen.  
   
   
       39 . A serology control according to  claim 33  where the derivative is serologically equivalent to A-antigen.  
   
   
       40 . A serology control according to  claim 33  where the modified cell surface epitope is serologically equivalent to the cell surface epitope of B-antigen.  
   
   
       41 . A serology control according to  claim 33  where the derivative is serologically equivalent to B-antigen.  
   
   
       42 . A serology control according to  claim 33  where the level of expression of modified cell surface epitope or derivative is serologically equivalent to the level of expression of A-/B-antigen by cells of a weak or poorly expressing ABO subgroup.  
   
   
       43 . A serology control according to  claim 33  where the level of modified cell surface epitope expression is serologically equivalent to the clinically significant threshold for expression of A-/B-antigen by an ABO blood group phenotype.  
   
   
       44 . A serology control according to  claim 33  where the H-antigen expressing cells are red blood cells.  
   
   
       45 . A serology control according to  claim 44  where the H-antigen expressing cells are human red blood cells.  
   
   
       46 . A serology control according to  claim 33  where the H-antigen expressing cells are animal cells wherein H-antigen has been incorporated into the cell membrane in vitro.  
   
   
       47 . A serology control according to  claim 33  where the level of expression of modified cell surface epitope or derivative is less than 5×10 5  copies per cell.  
   
   
       48 . A serology control according to  claim 33  where the level of expression of modified cell surface epitope or derivative is less than 1×10 5  copies per cell.  
   
   
       49 . A serology control according to  claim 33  where the level of expression of modified cell surface epitope or derivative is less than 2×10 4  copies per cell, or the serological equivalent thereof.  
   
   
       50 . A serology control according to  claim 33  where the level of expression of modified cell surface epitope or derivative is greater than 1×10 2  copies per cell.  
   
   
       51 . A serology control according to  claim 33  where the level of expression of modified cell surface epitope or derivative is greater than 1×10 3  copies per cell, or the serological equivalent thereof.  
   
   
       52 . A serology control according to  claim 33  where the modified cell surface epitope is serologically equivalent to the antigenic determinant of an immunodominant sugar.  
   
   
       53 . A serology control according to  claim 33  where the immunodominant sugar of an A-antigen or B-antigen.  
   
   
       54 . A serology control according to  claim 1  where the cells are in suspension.  
   
   
       55 . A serology control according to  claim 1  where the cells are localised to a surface.  
   
   
       56 . A serology control according to  claim 54  where the serology control contains a cell preservative such as Alsevers™, Cellstab™, Celpresol™.  
   
   
       57 . A serology control according to  claim 54  where the serology control contains clinically significant antibodies to provide an additional control characteristic.  
   
   
       58 . A serology control according to  claim 57  where the additional control characteristic is concurrent antibody control.  
   
   
       59 . A method for the determination of the sensitivity of a blood group type testing reagent including the steps of: 
 a. contacting the blood group testing reagent with a serology control according to  claim 1;  and    b. assessing the level of agglutination.    
   
   
       60 . A method according to  claim 59  where the assessing is by visual examination.  
   
   
       61 . A method according to  claim 59  including the step of determining the level of expression of modified cell surface epitope or derivative in the cell or cells of the serology control by reference to cells expressing known levels of antigen.  
   
   
       62 . A set or kit including two or more serology controls according to  claim 1 .  
   
   
       63 . A set or kit according to  claim 62  where the set or kit comprises serology controls including cells expressing the serological equivalent of group A and group B antigens.  
   
   
       64 . A set or kit according to  claim 62  where the set or kit comprises serology controls including red blood cells expressing the serological equivalent of group A, group B, Rh DCce (R1r) and Rh ce (rr) antigens.  
   
   
       65 . A set or kit according to  claim 63  where the expression is at a level substantially equivalent to a clinically significant threshold.